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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 Nov - 23 Dec 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
adopted in Feb 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Government of India, National Good Laboratory Practice (GLP) Compliance Monitoring Authority, India
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
TEST METHOD
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test substance by addressing the second molecular key event of the Adverse Outcome Pathway (AOP), activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™.
The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

TEST CELL LINE
- Type: KeratinoSens™ (Lot No. JRF/HaCaT/2016/01)
- Source: Givaudan, Switzerland
- Passage number: 21

CELL CULTURE CONDITIONS
- Type and identity of media:
Culture medium: Dulbecco`s Modified Eagle Medium (GlutaMAX™) supplemented with 9.1% fetal bovine calf serum and 500 µg/mL geneticin
Exposure medium: Dulbecco`s Modified Eagle Medium (GlutaMAX™) supplemented with 1% fetal bovine calf serum
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 1.0

TEST CONCENTRATIONS
0.98, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5, 125, 250, 500, 1000 and 2000 µM

CONTROLS
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
Positive control
- Substance: trans cinnamaldehyde
- Final concentration: 4 – 64 µM

EXPOSURE CONDITIONS
- Exposure duration: 48 ± 2 h
- Temperature (°C): 37 ± 1.0
- CO2 (%): 5.0 ± 1.0

NUMBER OF REPLICATIONS: three plates each in two independent experiments, 12-fold determination (test substance), 6-fold determination (control) and 5-fold determination (positive control) for each plate

DETERMINATION OF CELL VIABILITY
- Method: MTT assay
- MTT concentration: 5 mg/mL
- Incubation time: 4 h
- Device: plate reader
- Wavelength: 570 nm

DETERMINATION OF LUMINESCENCE
- Cell lysis reagent: Passive Lysis Buffer (Promega, Lot No. 0000174757)
- Luciferase Assay System: Luciferase Assay Kit (Promega, Lot No. 0000237533)
- Device: plate reader

Results and discussion

Positive control results:
The gene induction for the positive control trans cinnamaldehyde was found to be >1.5 at concentrations of 32 µM and 64 µM in both repetitions. The EC1.5 value for the positive control was found to be 22.97 µM and 25.29 µM in Experiment I and II, respectively (within the acceptable range of 7.5 to 30 µM). The average gene induction for the positive control at 64 µM was found to be 3.31 and 2.72 (which lies within the acceptable range of 2 and 8) for Experiment I and II, respectively.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment I
Value:
2.82
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Test item concentration: 31.25 µM; IC50 = 25.56 µM
Key result
Parameter:
other: maximum luciferase activity induction (Imax)
Run / experiment:
Experiment II
Value:
2.17
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: Test item concentration: 31.25 µM; IC50 = 34.80 µM
Key result
Parameter:
other: EC1.5 (µM)
Run / experiment:
Mean of Experiment I and II
Value:
20.79
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
The EC1.5 mean values for the test substance was 20.79 µM, which was less than 1000 µM. The mean value of maximum induction (Imax) for the test substance was 2.50 at the test concentration of 31.25 µM, which was higher than 1.5 fold. The cellular viability was 87.64% and 41.76% at 15.63 and 31.25 µM, respectively, with induction of luciferase activity above 1.5 fold. Therefore, the evaluation criteria are met for the test substance.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control met the acceptance criteria and was correctly identified as non-sensitiser.
- Acceptance criteria met for positive control: The positive control met the acceptance criteria and was correctly identified as sensitiser.
- Acceptance criteria met for variability between replicate measurements: The coefficient of variation observed for the negative control during Experiment I and II were 14.65% and 15.30%, respectively, which was below 20%. Since variation between the replicates was less than 20%, results of this run were considered as valid.

Any other information on results incl. tables

Table 1: Summary of Mean Induction (Experiment I and II)

Concentration

(µM)

Test item

Mean

SD

0.98

1.07

0.17

1.95

1.05

0.26

3.91

1.09

0.14

7.81

1.15

0.00

15.63

1.04

0.12

31.25

2.50

0.46

62.5

1.24

0.23

125

0.00

0.00

250

-0.01

0.00

500

0.00

0.00

1000

0.00

0.00

2000

0.00

0.00

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitising potential based on the key event "activation of keratinocytes"
Conclusions:
Under the conditions of the test, it can be concluded, that the test substance is a sensitiser in KeratinoSens Assay. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.